The therapeutic use of hematopoietic stem cell (HSC) transplantation for inherited or acquired blood diseases is often restricted due to the difficulty with finding HLA-matched donors and immunologic intolerance between donor and recipient. Therefore, there is a critical need to identify new methods to generate high throughput, clinical-grade functional HSCs from human tissues. The objective of this application is to analyze novel cell-extrinsic mechanisms that stimulate the developmental transition from hemogenic endothelial cells to HSCs. We recently established that functional HSCs emerge in the cdh5-deleted zebrafish embryos, despite an impaired blood circulation. These findings led us to investigate shear-stress independent biomechanical mechanisms stimulating endothelial to HSC transition. Using echocardiography, micro-angiography, 3D Doppler, and confocal imaging, we demonstrate that heartbeat mediated pulse-pressure, and thus circumferential stretch stimulates HSC formation. Pharmacological inhibition of stretch-activated ion channels further reduced HSC formation. We, therefore, hypothesize that pulse-pressure mediated circumferential stretch stimulates the transition of endothelial cells to HSCs by stimulating stretch-activated ion channels. We will use advances in organ-in-a-dish and cell-engineering to recapitulate in situ circumferential stretch conditions on murine hemogenic endothelial cells. Since the silent-heart (tnnt2, sih)-silenced embryos lack heartbeat and blood circulation, we will also investigate if pharmacological stimulation of stretch-activated ion channels could rescue hematopoietic deficiency in the sih-silenced embryos. In addition, we will use transgenic zebrafish, ex vivo mouse embryo and explant culture methods, as well as confocal imaging to analyze how stimulation of stretch activated ion channels, Trpv4 and/or Piezo1, influences the emergence of functional HSCs. Our aims will establish a new conserved cell-extrinsic role and mechanisms of circumferential stretch during HSC formation. Identification of new factors stimulating hemogenic properties of endothelium would expedite the development of endothelial cells as a source of HSCs for the treatment of human blood diseases.